A number of patents and published applications disclose microfluidic devices that may be used to perform various chemical and biochemical analyses and syntheses. These disclosures include the following publications, all of which are incorporated by reference in their entirety for all purposes: U.S. Pat. No. 6,767,706 (“Integrated Active Flux Microfluidic Devices and Methods”), US20020127736A1 (“Microfluidic Devices And Methods Of Use”), US20020109114A1 (“Electrostatic Valves For Microfluidic Devices”), US20050145496A1 (“Thermal Reaction Device And Method For Using The Same”), and U.S. Pat. No. 6,729,352 (“Microfluidic Synthesis Devices And Methods”).
Additionally, the following applications and patents disclose relevant information pertinent to the present invention: U.S. non-provisional patent application Ser. No. 10/819,088, filed on Apr. 5, 2004 which claims priority to U.S. provisional patent application Ser. No. 60/460,634, filed on Apr. 3, 2003, both of which are incorporated by reference in their entirety for all purposes; U.S. Non-Provisional application Ser. No. 10/306,798, filed on Nov. 27, 2002, which claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 60/391,529, filed Jun. 24, 2002, and of U.S. Provisional Application No. 60/335,292, filed Nov. 30, 2001, each of which is incorporated by reference in their entirety for all purposes.
The general benefits of using microfluidic systems include a substantial reduction in time, cost and the space requirements for the devices utilized to conduct the analysis or synthesis.
Immunological methods are well known, and are performed routinely for diagnostic and research purposes. The problems with immunological methods are also well known, and are generally focused around increasing sensitivity while maintaining a high degree of specificity. Increasing sensitivity may be done by increasing the concentration of binding or target species, but increased concentration of reagents leads to an increased amount of non-specific binding of cross-reactive antibodies, leading to false positives. So there is a constant tension between the requirements for specificity and sensitivity.
Enzyme-linked Immunosorbent Assays (ELISAs) combine the specificity of antibodies with the sensitivity of simple enzyme assays, by using antibodies or antigens coupled to an easily-assayed enzyme. ELISAs can provide a useful measurement of antigen or antibody concentration. There are two main variations on this method: The ELISA can be used to detect the presence of antigens that are recognized by an antibody or it can be used to test for antibodies that recognize an antigen. An ELISA is generally a five-step procedure: 1) coat the microtiter plate wells with antigen; 2) block all unbound sites to prevent false positive results; 3) add antibody to the wells; 4) add anti-mouse IgG conjugated to an enzyme; 5) reaction of a substrate with the enzyme to produce a colored product, thus indicating a positive reaction. There are many different types of ELISAs.
One of the most useful of the immunoassays is the two antibody “sandwich” ELISA. This assay is used to determine the antigen concentration in unknown samples. If a purified antigen standard is available, the assay can determine the absolute amount of antigen in an unknown sample. The sandwich ELISA requires two antibodies that bind to epitopes that do not overlap on the antigen. This can be accomplished with either two monoclonal antibodies that recognize discrete sites or one batch of affinity-purified polyclonal antibodies. One antibody (the “capture” antibody) is purified and bound to a solid phase typically attached to the bottom of a plate well. Antigen is then added and allowed to complex with the bound antibody. Unbound products are then removed with a wash, and a labeled second antibody (the “detection” antibody) is allowed to bind to the antigen, thus completing the “sandwich”. The assay is then quantitated by measuring the amount of labeled second antibody bound to the matrix, through the use of a colorimetric substrate. Major advantages of this technique are that the antigen does not need to be purified prior to use, and that these assays are very specific. However, one disadvantage is that not all antibodies can be used. Monoclonal antibody combinations must be qualified as “matched pairs”, meaning that they can recognize separate epitopes on the antigen so they do not hinder each other's binding.
Unlike Western blots, which use precipitating substrates, ELISA procedures utilize substrates that produce soluble products. Ideally the enzyme substrates should be stable, safe and inexpensive. Popular enzymes are those that convert a colorless substrate to a colored product, e.g., p-nitrophenylphosphate (pNPP), which is converted to the yellow p-nitrophenol by alkaline phosphatase. Substrates used with peroxidase include 2,2′-azo-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD) and 3,3′5,5′-tetramethylbenzidine base (TMB), which yield green, orange and blue colors, respectively.
The sensitivity of the sandwich ELISA is dependent on four factors: (1) The number of molecules of the first antibody that are bound to the solid phase. (2) The avidity of the first antibody for the antigen. (3) The avidity of the second antibody for the antigen. (4) The specific activity of the second antibody.
When two “matched pair” antibodies are not available for a target, another option is the competitive ELISA. Another advantage to the competitive ELISA is that non-purified primary antibodies may be used. In order to utilize a competitive ELISA, one reagent must be conjugated to a detection enzyme, such as horseradish peroxidase. The enzyme may be linked to either the immunogen or the primary antibody. A labeled immunogen is commonly used as the competitor.
In use, an unlabeled purified primary antibody is coated onto the wells of a 96 well microtiter plate. This primary antibody is then incubated with unlabeled standards and unknowns. After this reaction is allowed to go to equilibrium, conjugated immunogen is added. This conjugate will bind to the primary antibody wherever its binding sites are not already occupied by unlabeled immunogen. Thus, the more immunogen in the sample or standard, the lower the amount of conjugated immunogen bound. The plate is then developed with substrate and color change is measured.
Conventional ELISAs generally provide a plate with a number of reaction wells onto which a number of primary antibodies have been pre-spotted. The wells are then flooded with the antigen to be detected which binds via a first epitope to the primary antibody. The secondary labeled antibody is then flooded onto the plate and binds specifically to a second epitope of the antigen. A signal is then produced and detected by use of a colorimetric substrate.
A major problem with current antibody systems is caused by cross-talk between the antibodies. This problem is inherent in a system that requires exposure of the primary antibody to a cocktail of polyclonal secondary antibodies, some of which may bind non-specifically not to the bound antigen, but to the primary antibody, creating false positives.